The objective of this study is to quantify the hydrophobicity of biological substances and their structural elements for use in linear free energy relationships, LFER, between biological response and physico-chemical properties. High performance liquid chromatography with non-polar stationary phases of different surface geometries is employed to obtain experimental data which are evaluated by factor analysis. A large group of dihydrofolate reductase inhibitors whose biochemical activity is known, are subjected to chromatographic study and statistical analysis in order to find quantitative structure-activity relationships.